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Fig. 5. The PI3K/Akt pathway is required for IGF1-induced EFTFs. (A) Western blot showing that εmor injection blocked Akt phosphorylation induced by IGF1, but had no effect on Akt phosphorylation induced by P110. In addition, εmor had no effect on IGF1-induced ERK phosphorylation. εmor (10 ng) and RNA encoding IGF1 (2 ng) or P110 (1 ng) were injected sequentially, with εmor being injected at the 1-cell stage and RNAs being injected at the 4-cell stage. Animal caps were dissected at stage 8/9 and harvested at stage 13. (B) RT-PCR results showing LY294002, a PI3K inhibitor, blocked IGF1-induced expression of rx, lhx2, pax6, and six3, without affecting IGF1-induced otx2 and sox3 expressions in animal cap assay. IGF1 was injected as described above. Caps were dissected at late blastula stage and harvested at stage 13. Some caps were exposed to LY294002 from stage 9. (C) δp85 (2 ng) and dnAkt (2 ng) blocked IGF1 (2 ng) induced rx (upper panels), but not IGF1-induced otx2 (middle panels) and sox3 (lower panels) in whole embryos. One of the dorsal animal blastomeres was injected at the 8-cell stage. The right side was injected. |
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Fig. 6. The PI3K/Akt pathway is required for eye induction. (A) Injection of δp85 (2 ng) or dnAkt (2 ng) into dorsal animal blastomeres at the 8-cell stage blocked eye formation. (B) δp85 and dnAkt blocked the expression of rx (upper panels) and lhx2 (lower panels) at stage 14. (C) Injection of δp85 or dnAkt into A1 blastomere at the 32-cell stage impaired eye formation on the injected side (left) when embryos reached tadpole stage. Left side was injected. (D) Injection of δp85 (middle panels) or dnAkt (lower panels) into A1 blastomere at the 32-cell stage reduced the expression of rx, but not myoD, otx2, and sox3. n-β-gal was used as a lineage tracer. |
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Fig. 1. Dose-dependent effects of B56ε depletion on eye development. (A) Whole mount in situ hybridization showing the expression of rx (upper panels) and six3 (lower panels) in stage 20 control embryos, 2.5 ng εmor injected embryos, 5 ng εmor injected embryos, and 7.5 ng εmor injected embryos. εmor was injected into two dorsal animal blastomeres at the 8-cell stage. (B) Western blot showing the effects of εmor and 5mis on the translation of B56ε-FLAG. Myc-EGFP was used as a control for injection and loading. Morpholinos (5 ng) were injected into one of the dorsal animal blastomere at the 8-cell stage. Subsequently, a mixture of B56ε (200 pg) and Myc-EGFP (50 pg) was injected into the same blastomere. Injected embryos were harvested at the late gastrula stage. (C) Whole embryo phenotypes at the tadpole stage showing the effect of morpholinos injection on early embryonic development. Embryos were either uninjected (top), or bilaterally injected with εmor (5 ng, middle left; 7.5 ng, lower left), or injected with 5mis (5 ng, middle right; 7.5 ng, lower right) into both dorsal animal blastomeres at the 8-cell stage. (D) Whole mount in situ hybridization showing the expression of rx (upper panels) and six3 (lower panels) in stage 18 control embryos, 5 ng of 5mis injected embryos, and 7.5 ng of 5mis injected embryos. 5mis was injected into two dorsal animal blastomeres at the 8-cell stage. Note that less than 30% of embryos injected with 7.5 ng of 5mis exhibited fused rx or six3 expression at this stage. The rest of embryos were normal (not shown). |
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Fig. 9. B56ε is required for the Hh pathway. (A) Whole mount in situ hybridization showing εmor (3.5 ng) injection blocked Shh (1 ng)-induced rx reduction (upper panels) and ptc-1 expression (lower panels) in whole embryos. Embryos were bilaterally injected at the 8-cell stage. (B) RT-PCR showing εmor (10 ng) injection blocked ptc-1 and foxA2 induced by shh (1 ng), smo-M2 (2.5 ng), and gli1 (0.5 ng) in neuralized animal caps. εmor and RNAs were injected sequentially at the 1-cell stage. Animal caps were dissected at stage 8/9 and harvested at stage 14. (C) RT-PCR showing εmor injection (10 ng) blocked ptc-1 expression induced by wild-type Gli1 (500 pg), but not by Gli-VP (50 pg and 100 pg). Embryos were injected and manipulated as described above. (D) Western blot showing overexpression of Shh inhibited the processing of overexpressed Myc-Gli3 (0.5 ng) in animal caps. The effect of Shh (1 ng) on Myc-Gli3 processing was not affected by εmor (10 ng) injection. Embryos were injected and manipulated as described above, except that caps were harvested at stage 12. (E) Co-IP results showing overexpression of Shh (1 ng) dissociated complex formation between FLAG-Cos2 (1 ng) and Myc-Gli1 (1 ng) in animal caps. Injection of εmor (10 ng) did not affect this complex dissociation induced by Shh. Embryos were injected and manipulated as described above. (F) Whole mount in situ hybridization showing the expression of rx in (from left to right) a control embryo, an embryo injected with εmor (3.5 ng), an embryo injected with Gli-VP (100 pg), and an embryo injected with Gli-VP and εmor. Embryos were bilaterally injected at the 8-cell stage. |